MDDC - 1047 UNITED STATES ATOMIC ENERGY COMMISSION SYNTHESIS OF CHLORACETIC ACID, lODOACETIC ACID AND GLYCINE, LABELED WITH C-14 IN THE CARBOXYL GROUP by Rosemarie Cstwald University of California This document consists of 4 pages. Date of Manuscript: May 5, 1947 Date Declassified: June 24, 1947 This document is issued for official use. Its issuance does not constitute authority to declassify copies or versions of the same or similar content and title and by the same author (s). Technical Information Division, Oak Ridge Directed Operations Oak Ridge, Tennessee SYNTHESIS OF CHLORACETIC ACID, lODOACE TIC ACID AND GLYCINE, LABELED WITH C-14 IN THE CARBOXYL GROUP By Rostniarie Oslwiild In order to investigate protein metabolism with the helj) of radioactive c arljon, it ^;(•enu'd de- sirable to prepare some of the more important amino acids labeled with C'^. Carboxyl -labeled glycine and the necessary intermediate product carboxyl-labeled chloracdic acid were prepared. (Glycerine labeled with C'^ has been prepared oy Olsen, Hcmingw.iy, and Nier,' Lorber and Olsen, ^ and Sakami, Evans, and Gurin.^) Carboxyl -labeled iodoacetic acid was prepared to study its reactions with certain proteins, containing SH-groups. The methods and techniques employed were chosen with the object of obtaining lushest possible yields. Therefore, any unnecessary steps in the preparation of the comixjunds were avoided and t'^chniques suitable for handling small amounts of material were used throughout. EXPERIMENTAL PART Chloracetic Acid In order to generate anhydrous acetic acid from sodium acetat , a platinum boat, charged with 0.7049 g carboxyl -labered anhydrous sodium acetate* and 0.7032 g inactive sodium acetate tryhidrate was placed in a horizontal glass tube and was evacuated to 3 x 10"^ mm Hg and kept at that pressure for 24 hours at room temperature. Under these conditions the sodium acetate had been shown to be anhydrous in separate experiments. The glass tube was connected to a drying train of concentrated sulphuric acid, Drierite, and phosphorus pentoxide, which was swept with nitrogen while the salt was heated to its melting point. After the mixture was cooled, the other end of the tube was connected to a train of three traps, each of which was cooled with an isopropyl alcohol -dry ice mi.xture; the last trap was protected with a calcium chloride tube. Gaseous hydrogen chloride was passed slowly through the train, the tube was gradually heated, and the liberated acetic acid distilled into the traps. When the reaction was complete, the three traps were connected to the vacuum line and the contents were distilled Into a small reaction vessel (see Figure 1), which was cooled with liquid nitrogen. The product consisted of about 85-90f?) acetic acid, 10-15'(- water and a considerable amount of gaseous hydrogen chloride, as was determined by titration in separate runs. A low-temperature condenser, cooled with isopropyl alcohol-dry ice and protected with a calcium chloride tube, was attached to the reaction vessel which was then allowed to come to room temperature; 0.65 g of inactive acetic anhydride was added and the mixture was rcfluxed for one half hour to destroy the ICflb of water which was present. No attempt was made to determine the actual yield of acetic acid, a mixture of 0.02 g of iodine, 0.04 g of phosphorus, and 0.88 g phosphorus pentoxide were added, ^ and dry chlorine was passed through the system at reflux temperature for two and one half hours. The condenser was a normal Liebig type with a low-temperature condenser *Carboxyl -labeled sodium acetate was prepared by R. Lemmon from methyl-iodide and C by the Grignard reaction. MDDC - 1047 I 1 2] MDDC - 1047 sealed onto the top (see Figure 1). The whole apparatus was evacuated to 3 x 10"' mm and all the material from the condenser and the gas-inlet tube was distilled back into the reaction vessel, which was cooled in liquid nitrogen. The chloracetic acid was then purified by fractional sublima- tion onto a cold-finger condenser, filled with.powdered dry ice. All parts of the apparatus were connected by ground-glass joints. ISOPROPYL ALCOHOL DRY ICE . CHLORINATION REACTION VESSEL \^ Figure 1. The yield of pure product was 1.52 g (67''n, based on 1.129 g anhydrous sodium acetate) mp, 60°. Previous runs with inactive material gave yields of SS-yC^o. The sodium acetate used for the synthesis had a specific activity of 1.6 x 10^ cts/min/mg of compound (3.3 x iCcts/min/mg or 1.9 X 10"' microcuries of barium carbonate). The total activity was 11.2 x 10' cts or 650 micro- curies. The chloracetic acid had a specific activity of 5 x IC cts/min/mg of compound MDDC - 1047 [ 3 2 (1.19 X 10 cts/min/mg or 7.0 x 10" microcuries of barium carbonate) and a total activity of 7.6 X lO' cts or 440 microcuries. The activity recovered from mother liquors, washings, etc., was 1.7 X lO' cts or 100 microcuries. The specific activities were determined by combustion of the sample with an appropriate amount of benzoic acid as carrier. The carbon dioxide was precipitated as barium carbonate and was counted with a Geiger-Mueller counter with a geometry of 12.9 disintegrations per count. ^ lodoacetic Acid A mixture of 0.2408 g of carboxyl-labeled and 0.3 g of inactive chloracetic acid was added to a solution of 1.5 g of sodium iodide in 20 cc of dry acetone. After the mixture was kept at room temperature for 20 hours, the white precipitate of sodium chloride was filtered, washed with dry acetone, and the combined brown solutions were brought to a pH of 6.8 by dropwise addition of 40% sodium hydroxide, under vigorous stirring. The precipitate which formed was filtered immediately, washed with dry acetone, ice-cold absolute alcohol and ether. ° In trial runs, 85-90% yields of sodium iodoacetate were obtained. The identity and purity were established by titration of a con- centrated solution of the salt with 2N hydrochloric acid, using a pH meter with micro electrodes. The product was 98-99% pure. With active chloracetic acid a yield of 500 mg or 45% was obtained. The total activity of the chloracetic acid used was 1.2 x 10' cts or 70.4 microcuries. The sodium iodoacetate had a specific activity of 1.1 x 10* cts/min/mg of compound (5.6 x 10' cts or 3.3 X 10 microcuries) and a total activity of 5.5 x 10° cts or 32.2 microcuries. The activity re- covered from the sodium chloride precipitate, mother liquors, etc., was 3.4 x 10' cts or 2 micro- curies. The balance was lost, probably due to the volatility of iodine -containing compounds which were formed by decomposition in the strongly alkaline mother liquors. The sodium chloride precipitate, which weighed 600 mg instead of 330 mg expected from the re- action, was burned. It had a specific activity of 5000 cts/min/mg and a total activity of 3 x 10° cts or 17.6 microcuries. Efforts to recover any of the 280 mg sodium iodoacetate to which this activity corresponds, were unsuccessful. Glycine A mixture of 3.2 g of powdered ammonium carbonate, 10 cc of concentrated ammonia, and 4 cc of water, was heated in a small three-neck flask, which was fitted through ground-glass joints to a pressure -equalized dropping funnel, a Liebig condenser, and a thermometer. Aft°r the salt had dissolved, 1.014 g of carboxyl-labeled chloracetic acid in 3 cc of water was added dropwise through the dropping funnel, at such a rate that the temperature of the solution did not rise above 60°. The mixture was held at 60° for six hours and was then allowed to stand for twelve hours at room tem- perature. The solution was then concentrated until its temperature reached 112°. The distillate showed only very slight radioactivity. The yellowish solution was cooled to 70°, and 15 cc of absolute methanol was added slowly with agitation. The mixture was cooled in a refrigerator for one hour. The precipitate was filtered and washed with methanol and ether.' The yield of pure white crystals, which showed no trace of chloride ion was 0.54 g or 70% (melting point 225°, dec). Upon concentration, the mother liquor gave 0.08 g of glycine which in- creased the yield to 0.62 g or 79%. The chloracetic acid used had a total activity of 5.1 x 10' cts or 299 microcuries. The glycine had a specific activity of 6.3 x lO* cts/min/mg of compound (1.19 cts/min/mg of barium carbonate or 3.3 X 10 microcuries) and a total activity of 3.9 x 10 cts or 229 microcuries. From distillates, mother liquors, etc., 8.0 x 10° cts or 50.4 microcuries were recovered. SUMMARY 1. Carboxyl-labeled chloracetic acid was prepared by passing a stream of dry chlorine through a mixture of anhydrous carboxyl-labeled acetic acid, using a mixture of iodine, phosphorus, and 4] MDDC - 1047 phosphorus pentoxide as catalyst. The carboxyl -labeled, anhydrous acetic acid was liberated from carboxyl -labeled sodium acetate by heating the dry salt in a stream of dry gaseous hydrogen chlo- ride and distilling the liberated acetic acid. 2. Carboxyl -labeled iodoacetic acid was prepared from carboxyl -labeled chloracetic acid by allowing a mixture of chloracetic acid and sodium iodide in dry acetone to stand for twenty hours at room temperature. The sodium chloride was filtered, and the sodium iodoacetate was precipitated by adjusting the solution to a pH of 6.8 with 40% sodium hydroxide. 3. Carboxyl -labeled glycine was prepared by heating a mixture of carboxyl -labeled chloracetic acid, ammonia, and ammonium carbonate in the proportion of 1 : 15 : 5 for six hours to 60° and precipitating the glycine by addition of absolute methanol, after the solution had been concentrated. The author wishes to thank Professor M. Calvin for his advice in this work. REFERENCES 1. Olsen, Hemingway and Nier, J. Biol. Chem. 148: 611 (1943). 2. Lorber and Olsen, Proc. Soc. Exptl. Biol. Med. 61: 227 (1946). 3. Sakami. Evans, and Gurin, Report lB-40 Isotopes Br., Res. Div., Manhattan District. 4. Yorst Brueckner, Z. angew. Ch. 40: 973 and 41: 228. 5. Dauben, Reid, and Yankwich, paper in preparation; Yankwich, Norris, and Huston, paper in preparation. 6. Goldberg, Leon, Science 98: 386 (1943). 7. Cheronis, N. D. and K. H. Spitzmueller, J". Org. Ch. 6: 349, (1941); Orten, J. M. and R. M. HUl, Org. Synthesis Coll. 1: 300. UNIVERSITY OF FLORIDA 3 1262 08909 7967